Health care remains at the foremost frontiers for scientific research. The need to discover and develop cost-effective and safer medications is ever increasing. The ability to accurately model the cellular organization within a particular tissue or organ is of paramount importance. A close replica of the in vivo system to in vitro would require cell growth in three dimensions (3D). The “cross-talk” achieved between the cells in a 3D cell culture in vitro is a close mimic of cell growth under physiological conditions. Indeed, 3D cell culture have assumed significance in efforts directed towards regenerative medicine, better understanding of chronic diseases and providing superior in vitro model system for screening drugs and toxicological assays. Its emergence is thus being aptly touted as “biology's new dimension”.
Intense research efforts are on to identify and develop “factors and scaffolds” that would favor 3D cell growth in vitro. The cells under physiological conditions not only “cross-talk” amongst themselves but also interact with the cellular microenvironment, the extra-cellular matrix (ECM), with which they reside. The ECM provides structural support to the cells and also contributes to signaling and directing cell fate. Mostly, the ECM is composed of glycosaminoglycans and fibrous proteins such as collagen, elastin, laminin and fibronectin self assembled into nanofibrillar network. An ideal scaffold for 3D cell growth should be able to mimic the structural component of native ECM, support cell growth and maintenance, have the right sized network of interconnected pores for efficient cell migration and transfer of nutrients to the cells. In essence, the mechanical and chemical properties of the scaffold should lead to cellular function as in the native state.
Hydrogels, both of synthetic and natural origin have emerged as suitable scaffolds for 3D cell culture. The network of interconnected pores in hydrogels allows for retention of a large amount of biological fluid, facilitates transport of oxygen, nutrients and waste. Furthermore, most hydrogels can be formed under mild cytocompatible conditions and the biological properties can be modulated by surface chemistry. Engineered hydrogels with modified mechanical, chemical and biological properties have the potential to mimic the ECM and thus establish their utility in 3D cell culture. Commercial products for 3D cell culturing are for example PURAMATRIX™ (3DM Inc.) and MATRIGEL® (BD Biosciences). PURAMATRIX™ is a hydrogel of self-assembled peptide nanofibers which resembles the structure of natural fibrillar collagen in ECM with fiber diameter 5-10 nm. It has also high water content, typically 99.5%. U.S. Pat. No. 7,449,180 and WO 2004/007683 disclose peptide hydrogels. MATRIGEL® is gelatinous protein mixture secreted by mouse tumor cells. The mixture resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for cell culture. MAXGEL™ ECM Matrix (Sigma-Aldrich), which includes a mixture of human ECM components, forms a gel in ambient temperature.
Bacterial cellulose has been used in wound healing membranes and as a scaffold in cell culture. The limitation in the use of bacterial cellulose in cell culture is the inherent structure of the fermented material; upon cultivation, BC is formed as very tight membranes in air water interphase in the fermenter. The formed membranes are too tight for many 3D cell culturing tasks and various modifications are needed to improve the porosity, which is needed for cell penetration and formation of cell clusters.
Hydrogel materials are also widely used in other types of culturing tasks where hydrophilic supporting material is needed, for example agar type hydrocolloids are widely used in plant cell, bacterial, and fungi culturing for various microbiological purposes.
U.S. Pat. No. 5,254,471 discloses a carrier for culturing cells made of ultra fine fibers. WO 2009/126980 discloses cellulose-based hydrogel, which contains cellulose exhibiting an average degree of polymerization of 150-6200.
The solutions of the prior art have been found to be rather unsatisfactory in cell culture. All the present 2D and 3D cell culture methods and matrices require the use of animal based chemicals or compounds on the biomaterial media in order to cells to be maintained and multiplied. Maintenance of stem cells is especially demanding and there exists no simple solutions for matrix used with cell culture media which would keep the stem cells alive. The presence of animal based compounds in cell culture environment generates a serious risk of immunoreactions, and different types of toxicity issues, which finally will kill the cultured cells. Cell culture matrices containing animal-based additives are not suitable for use with stem cells, especially, if stem cells are to be used for tissue transplantation and tissue technology (engineering). Furthermore, many of the polymers proposed for use in the cell culture media do not tolerate a physiological temperature or are toxic for cells.